Genomic imprinting is an epigenetic mechanism affecting a small group of genes in mammals and that results in monoallelic parental-specific expression. Many imprinted genes code for regulators of proliferation or differentiation, so alterations in their regulation can play a role in developmental abnormalities and cancer. Therefore, gaining a full comprehension of the mechanisms by which imprinting is regulated is crucial to understanding normal embryonic development and the pathogenesis of many human diseases. H19 and Igf2 are two linked genes with a similar temporal and tissue- specific pattern of expression and are imprinted. Hl9 is expressed from the maternal allele, whereas Igf2 is expressed paternally. The coordinate regulation of imprinting at these loci depends on a 2 kb differentially methylated domain (DMD) that lies upstream of the H19 gene. This DMD contains four GC-rich repeats that are highly conserved between the mouse, rat and human sequences, a fact that suggests that they are an important feature for the regulation of the domain. The four repeats exhibit methylation-dependent boundary activity in vitro. The first goal of these studies is to determine the requirement of the four CG-rich 21- by repeats within the DMD for the establishment of imprinting. The second aim is to determine whether methylation of specific CG dinucleotides within these repeats is a signal for the parental identity of the alleles. Both aims will be accomplished by gene targeting at the endogenous locus. These experiments will lead to enhanced understanding of the molecular mechanisms underlying imprinting.